Mineralization and cellular patterning on biomaterial surfaces

ABSTRACT

Disclosed are advantageous methods for patterning and/or mineralizing biomaterial surfaces. The techniques described are particularly useful for generating three-dimensional or contoured bioimplant materials with patterned surfaces or patterned, mineralized surfaces. Also provided are various methods of using the mineralized and/or patterned biomaterials in tissue engineering, such as bone tissue engineering, providing more control over ongoing biological processes, such as mineralization, growth factor release, cellular attachment and tissue growth.

BACKGROUND OF THE INVENTION

[0001] The present application claims priority to second U.S.provisional application Ser. No. 60/167,289, filed Nov. 24, 1999, whichclaims priority to first U.S. provisional application Ser. No.60/125,118, filed Mar. 19, 1999, the entire text and figures of whichapplications are incorporated herein by reference without disclaimer.The U.S. Government owns rights in the present invention pursuant togrant numbers R01 DE13033 and T32 GM 08353 from the National Institutesof Health.

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the diverse fields oflithography, chemistry, biomaterials and tissue engineering. Moreparticularly, it concerns the patterning and/or mineralization ofbiopolymers. These methods provided are particularly suited to thegeneration of surface-modified three-dimensional biomaterials for use incell culture, transplantation and tissue engineering.

[0004] 2. Description of Related Art

[0005] Many biomedical procedures require the provision of healthytissue to counteract the disease process or trauma being treated. Thiswork is often hampered by the tremendous shortage of tissues availablefor transplantation and/or grafting. Tissue engineering may ultimatelyprovide alternatives to whole organ or tissue transplantation.

[0006] In order to generate engineered tissues, various combinations ofbiomaterials and living cells are currently being investigated. Althoughattention is often focused on the cellular aspects of the engineeringprocess, the design characteristics of the biomaterials also constitutea major challenge in this field.

[0007] In recent years, the ability to regenerate tissues and to controlthe properties of the regenerated tissue have been investigated bytrying to specifically tune the mechanical or chemical properties of thebiomaterial scaffold (Kim et al., 1997; Kohn et al. 1997). The majorityof this work has involved the incorporation of chemical factors into thematerial during processing, or the tuning of mechanical properties byaltering the constituents of the material.

[0008] The foregoing methods have been used in an attempt to utilizechemical or mechanical signaling to affect changes in the proliferationand/or differentiation of cells during tissue regeneration. Despite suchefforts, there remains in the art a need for improved biomaterials,particularly those with a better capacity to support complex tissuegrowth in vitro (in cell culture) and in vivo (upon implantation).

SUMMARY OF THE INVENTION

[0009] The present invention overcomes various drawbacks in the art byproviding a range of improved methods, compositions and devices for usein cell culture, cell transplantation and tissue engineering. Themethods, compositions and apparatus of the invention involve patternedand/or mineralized biomaterial surfaces. The techniques and productsprovided are particularly useful for generating three-dimensional orcontoured bioimplant materials with modified surface features and forgenerating biomaterials incorporating bioactive factors and/or cells.The various methods of using the mineralized and/or patternedbiomaterials in tissue engineering, including bone tissue engineeringand vascularization, thus provide more control over the biologicalprocesses.

[0010] Unifying aspects of the invention involve the surfacemodification, functionalization or treatment of biocompatible materials.Such modifications, functionalizations or treatment methods arepreferably used to create reactive surfaces that may be furthermanipulated, e.g., patterned and/or mineralized. The patterned and/ormineralized biocompatible materials have a variety of uses, both invitro and in vivo.

[0011] A first general aspect of the present invention concerns thepatterned treatment of polymer biomaterial surfaces using a unique“diffraction lithography” process. Prior lithographic methods of surfacepatterning have been limited to flat, two dimensional surfaces, which isa significant limitation overcome by the methods provided herein. Thepresent invention is thus applicable to surface patterning on complexthree dimensional biomaterials with surface contours.

[0012] The development of these aspects of the overall invention isparticularly surprising as it provides patterns of sufficient resolutionto be useful in biological embodiments. Further advantages of theinvention over the methods of the prior art include the readyincorporation of biologically active components into the patternedbiomaterials and the reduced risk of contamination. Other significantfeatures of the invention are the cost-effectiveness and labor-savingnature of the techniques.

[0013] A second general aspect of the invention involves the surfacetreatment or functionalization of a biocompatible material, preferably aporous, degradable polymer, such as a film or sponge, to spur nucleationand growth of an extended mineral layer on the surface. Such treatmentcan be controlled to provide a homogeneous surface mineral layer or apatterned mineral layer, such as islands of minerals. Each of suchextended mineral layers allow the growth of continuous bone-like minerallayers, even on inner pore surfaces of polymer scaffolds.

[0014] Such extensively mineralized, patterned mineralized and/orhypermineralized polymers of the invention have advantageous uses inbone tissue engineering and regeneration and tissue vascularization. Theformation of extended mineral islands and/or substantially homogeneous,“continuous” mineral layers, particularly those on the inner poresurfaces of three dimensional matrices, is advantageous as it can beachieved simply (a one step incubation), quickly (about five days), atroom temperature, without leading to an appreciable decrease in totalscaffold porosity or pore size, and is amenable to further incorporationof bioactive substances.

[0015] The further incorporation of bioactive substances is exemplifiedby the formation and use of polymers, preferably, biodegradablepolymers, that are both mineralized and provide for the sustainedrelease of bioactive factors, such as protein growth factors. In theseaspects of the invention, the type of mineral layer may be controlled byaltering the molecular weight of the polymer; the composition of thepolymer; the processing technique (solvent casting, heat pressing, gasfoaming) used to prepare the polymer; the type and/or density of defectson the polymer surface; and/or by varying the incubation time.

[0016] The various improved biomaterials of the invention haveadvantageous uses in cell and tissue culture and engineering methods,both in vitro and in vivo. By way of example only, the present inventionprovides biomaterial methods and compositions with patterned mineralsurfaces for use in patterning bone cell adhesion.

[0017] Accordingly, the general methods of the invention are thosesuitable for the surface-modification of at least a first biocompatiblematerial or device, comprising:

[0018] (a) generating a patterned surface on a biocompatible material ordevice by a method comprising irradiating at least a firstphotosensitive surface of a biocompatible material or device withpre-patterned electromagnetic radiation, thereby generating a pattern onat least a first surface of the biocompatible material or device; and/or

[0019] (b) generating an extended mineralized surface on a biocompatiblematerial or device by a method comprising functionalizing at least afirst surface of a biocompatible material or device and contacting thefunctionalized surface with an amount of a mineral-containing solution,thereby generating extended mineralization on at least a first surfaceof the biocompatible material or device.

[0020] The irradiation, lithographic or diffractive lithography methodsgenerally comprise generating a patterned surface on a biocompatiblematerial by a method comprising functionalizing at least a firstphotosensitive surface of a biocompatible material by irradiating thephotosensitive surface with an amount of pre-patterned electromagneticradiation effective to generate a patterned biocompatible materialcomprising a pattern on at least a first surface of the biocompatiblematerial. In these methods, the functionalized surface is preferablyfunctionalized to create a plurality of polar oxygen groups at thesurface, generally so that the functionalized surface can be furthermodified, e.g., with minerals, cells or the like.

[0021] It will thus be noted that the methods for generating a patternedsurface on a biomaterial or device, comprise “directly” applyingpre-patterned radiation to a photosensitive surface of a biomaterial ordevice. The “direct” application of the pre-patterned radiation is asignificant advantage as it occurs without the intervention of a “mask”,which is a significant drawback in contact lithography. The presentinvention thus provides “mask-less” or “naked” lithography forbiomaterial patterning in which pre-patterned radiation is impingingdirectly onto a photosensitive surface of a biomaterial in the absenceof an intervening mask.

[0022] “Electromagnetic radiation”, as used herein, includes all typesof radiation being electromagnetic in origin, i.e., being composed ofperpendicular electric and magnetic fields. The pre-patterned radiationfor use in the invention is preferably constructively and destructivelyinterfering electromagnetic radiation.

[0023] The present invention includes the use of all constructively anddestructively interfering radiation, such as constructive anddestructive interference based on amplitude, as well as phase hologramsthat rely on constructive and destructive interference based on phaseonly. One advantage of phase only holograms is that more light getsthrough, and a more complex pattern can be formed. However, the use ofdiffraction gratings to provide constructive and destructiveinterference based on amplitude is advantageous in construction andcost.

[0024] The pre-patterned radiation may be constructively anddestructively interfering radiation from any effective part of thevisible spectrum. Constructively and destructively interfering radiationin the UV, infrared and visible spectra are preferred examples, with UVand visible spectra being most preferred.

[0025] The pre-patterned, constructively and destructively interferingradiation may be generated by impinging monochromatic radiation on adiffractive optical element that converts the monochromatic radiationinto constructively and destructively interfering radiation.

[0026] The monochromatic radiation may be generated from any suitablesource. For example, one or more lasers or one or more mercury bulbs.The monochromatic radiation may be first generated from anelectromagnetic radiation source and then passed through a suitablefilter.

[0027] A wide range of diffractive optical elements may be used in theinvention. “Diffractive optical element” is a term that includesdiffraction gratings, holograms, and other pattern generators. There isvirtually no limitation to these aspects of the invention as anycomponent of the spectrum can be patterned by any type of opticalelement by varying the design of the optical element. For example, thereis a well defined relationship between the feature spacing in adiffraction pattern, and the spacing of the slits in the diffractionpattern plus the wavelength of the radiation. Thus, the slit widths canbe varied to create any pattern spacing with any wavelength ofradiation.

[0028] Therefore, one may use in the invention one or more diffractivelenses, deflector/array generators, hemispherical lenslets, kinoforms,diffraction gratings, fresnel microlenses and/or a phase-only holograms.Those of ordinary skill in the art will understand that a “diffractiongrating” actually produces an “interference pattern”, not a “diffractionpattern”, which is a matter of semantics resulting from the originalnaming of “diffraction gratings”.

[0029] The diffractive optical element(s) may also be fabricated fromany suitable material, such as a transparent polymer or glass. Examplesof transparent polymers are those selected from the group consisting ofa poly(methyl methacrylate), poly(styrene), and a high densitypoly(ethylene). Examples of diffraction gratings are those fabricatedfrom metal on glass, metal on polymer or metal with transmissionapertures (slits or holes). Other suitable diffractive optical elementsare those fabricated from fused silica or sapphire. The choice ofelement and matching of element to processing conditions will be routineto those of skill in the art.

[0030] Those of ordinary skill in the art will understand that UV lightis less suitable for use with cells. When using visible light, nocompromise of cell function is expected. Solely as a precaution, anupper limit may be about 6 W/cm² (Watts per square centimeter). Forinfrared light, a precautionary upper limit may be about 2.2 MW/cm²(Megawatts per square centimeter).

[0031] For use with proteins, a precautionary upper limit of UV may beabout 8 MW/cm² (Milliwatts per square centimeter). It is not believedthat an upper limit of intensity of visible light limits the applicationof the present invention to use with proteins. For use with proteins andcells, local heating during polymerization can be readily minimized,e.g., by using high molecular weight resins, and by decreasing totalpolymerization time.

[0032] Generating a pattern with pre-patterned electromagnetic radiationincludes the direct generation of a patterned surface that naturallyoccurs as a result of the electromagnetic radiation contacting thesurface of the biocompatible material. Therefore, the “photosensitivesurface” of the biocompatible material may simply be the “unmodified”biocompatible material surface. The “thereby generating” of the methodcan therefore be an inherent feature of the method.

[0033] “Thereby generating” may also include methods where theirradiated photosensitive surface is “developed” to provide thepatterned surface. Where the photosensitive surface has not been coatedwith any particular photosensitive material, the generation of thepatterned surface after irradiation preferably includes “developing” theirradiated photosensitive biomaterial to generate the patterned surface.“Developing” in this sense preferably involves washing or rinsing in asuitable liquid or solvent, such as water or an organic solvent.

[0034] The invention further includes more indirect methods ofgenerating the patterned surface, i.e., where the photosensitive surfaceto be irradiated is not the unmodified biomaterial surface. In suchmethods, the photosensitive surface is prepared by applying aphotosensitive composition, admixture, combination, coating or layer toat least a first surface of the biocompatible material.

[0035] The photosensitive composition may be applied to at least a firstsurface of the biocompatible material by contacting the biocompatiblematerial with a formulation of the photosensitive composition in avolatile solvent and evaporating the solvent to coat the photosensitivecomposition onto the at least a first surface. The photosensitivecomposition may also be applied to at least a first surface of thebiocompatible material by contacting the biocompatible material with aformulation of the photosensitive composition in an aqueous or colloidalsolution to adsorb the photosensitive composition onto the at least afirst surface.

[0036] The invention thus comprises:

[0037] (a) applying a photosensitive layer to at least a first surfaceof a biomaterial;

[0038] (b) creating pre-patterned radiation;

[0039] (c) irradiating the photosensitive layer with the pre-patternedradiation to form an irradiated layer; and

[0040] (d) developing the irradiated layer to generate a pattern on theat least a first surface of the biomaterial.

[0041] The invention further comprises:

[0042] (a) applying a photosensitive layer to at least a first surfaceof a biomaterial;

[0043] (b) obtaining a monochromatic radiation source;

[0044] (c) impinging the monochromatic radiation source on an elementthat converts the monochromatic radiation into patterned radiation;

[0045] (d) irradiating the photosensitive layer with the patternedradiation to form an irradiated layer; and

[0046] (e) developing the irradiated layer to generate a pattern on theat least a first surface of the biomaterial.

[0047] The invention still further comprises:

[0048] (a) applying a photosensitive layer to at least a first surfaceof a biomaterial;

[0049] (b) obtaining a monochromatic radiation source;

[0050] (c) transmitting the monochromatic radiation source through anelement that transforms the monochromatic radiation into patternedradiation;

[0051] (d) impinging the transmitted patterned radiation onto thephotosensitive layer of the biomaterial to form an irradiated layer; and

[0052] (e) developing the irradiated layer to generate a pattern on theat least a first surface of the biomaterial.

[0053] Any one of a wide variety of photosensitive compositions may beused. Such compositions generally comprise a combined effective amountof at least a first photoinitiator and at least a first polymerizablecomponent.

[0054] Suitable photosensitive compositions may comprise apolymerization-initiating amount of at least a first UV-excitablephotoinitiator, such as a UV-excitable photoinitiator selected from thegroup consisting of a benzoin derivative, benzil ketal,hydroxyalkylphenone, alpha-amino ketone, acylphosphine oxide,benzophenone derivative and a thioxanthone derivative.

[0055] Other photosensitive compositions may comprise apolymerization-initiating amount of at least a first visiblelight-excitable photoinitiator, such as a visible light-excitablephotoinitiator selected from the group consisting of eosin, methyleneblue, rose bengal, dialkylphenacylsulfonium butyltriphenylborate, afluorinated diaryltitanocene, a cyanine, a cyanine borate, aketocoumarin and a fluorone dye. These photosensitive compositions mayfurther comprise a co-initiating amount of at least a first co-initiatoror accelerator, such as a co-initiator or accelerator selected from thegroup consisting of a tertiary amine, peroxide, organotin compound,borate salt and an imidazole.

[0056] The choice of components for use in the photosensitivecompositions will be straightforward to those of skill in the art.Essentially any photoinitiator or initiator system and any “resin”(types of components or monomers to be photopolymerized) can becombined.

[0057] The choice of resin is therefore wide. For example, a suitable“multifunctional acrylate” is any monomer that can be acrylated.

[0058] The resin components are used in photopolymerizable amounts, suchas photopolymerizable amounts of at least a first monomeric, oligomericor polymeric polymerizable component. Suitable polymerizable monomersinclude those selected from the group consisting of an unsaturatedfumaric polyester, maleic polyester, styrene, a multifunctional acrylatemonomer, an epoxide and a vinyl ether.

[0059] One currently preferred photosensitive composition comprises acombined effective amount of an eosin photoinitiator, a poly(ethyleneglycol) diacrylate polymerizable component and a triethanolamineaccelerator.

[0060] The methods of the invention produce patterns with a resolutionof between about 1 μM and about 500 μM; of between about 1 μM and about100 μM; of between about 10 μM and about 100 μM; of between about 1 μMand about 10 μM; and of between about 10 μM and about 20 μM. These arehighly suitable for biomedical embodiments, although substantiallyunsuitable for microelectronic embodiments, as a single cell is in the10 μM to 20 μM range. Patterns with a resolution of about 0.5, 0.75, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 50, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, 550 or about 600 μM or so can be produced andused to advantage.

[0061] An advantage of the invention is that the entire processes can becarried out at biocompatible temperatures. For example, a biocompatiblematerial can be maintained on a temperature-controlled support duringirradiation.

[0062] The biocompatible materials, either before, during or afterpatterning, may be contacted with an amount of a mineral-containingsolution effective to generate some, moderate, or preferably extendedmineralization on at least a first surface of the biocompatiblematerial. Such methods link to the mineralization methods and comprisecontacting with a mineral-containing solution prior.

[0063] Preferably, the biocompatible material is contacted with themineral-containing solution during or subsequent to the generation ofthe patterned surface, thereby forming a mineralized biocompatiblematerial comprising a pattern of minerals on least a first surface.Furthermore, at least a first mineral-adherent biological cell may besubsequently bound to the mineralized biocompatible material to form apattern of biological cells on least a first surface of thebiocompatible material.

[0064] Both the mineral adherence and/or cell adherence may be carriedout by exposure of the biocompatible material and/or mineralizedbiocompatible material to a population of minerals and/or cells eitherin vitro or in vivo. Sequential or simultaneous exposure may be used.

[0065] In the mineralization methods of the invention, one generates anextended mineralized surface on a biocompatible material by a methodcomprising functionalizing at least a first surface of a biocompatiblematerial to create a plurality of polar oxygen groups at afunctionalized surface and contacting the functionalized surface with anamount of a mineral-containing solution effective to generate extendedmineralization on the at least a first surface of the biocompatiblematerial.

[0066] The methods may comprise generating the functionalized surface byexposing at least a first surface of the biocompatible material to afunctionalizing pre-treatment prior to contact with themineral-containing solution. Effective functionalizing pre-treatmentsinclude exposure to an effective amount of electromagnetic radiation,such as UV radiation; exposure to an effective amount of electron beam(e-beam) irradiation; and exposure to functionalizing biocompatiblechemicals, such as an effective amount of a NaOH solution.

[0067] The methods also comprise one-step methods wherein thefunctionalized surface is generated during the contact with themineral-containing solution. Such single step methods for forming amineralized biomaterial that comprises an extended mineral coating on abiomaterial surface comprise incubating a mineralizable biomaterial withan amount of a mineral-containing solution, such as an aqueous mineralsolution, effective to generate a functionalized biomaterial surfaceupon which an extended mineral coating forms during the incubation.These methods are preferred for use with polymer or copolymerbiomaterials, such as polylactic acid (PLA) polymer, polyglycolic acid(PGA) polymer or polylactic-co-glycolic acid (PLG) copolymerbiomaterials.

[0068] Any mineralization method, whether pre-patterned or not, may usea mineral-containing solution that comprises calcium, wherein theresultant mineralization or extended mineralization comprises anextended calcium coating. Mineral-containing solutions may also compriseat least a first and second mineral, wherein the resultantmineralization or extended mineralization comprises a mixture of thefirst and second minerals. Mineral-containing solutions may furthercomprise a plurality of distinct minerals, wherein the resultantmineralization or extended mineralization comprises a heterogeneouspolymineralized coating.

[0069] The methods are controllable to provide mineralization, extendedmineralization, patterned mineralization, extended patternedmineralization, substantially homogeneous mineral coatings,hypermineralized portions or regions, inner pore surfaces of porousmaterials wherein a mineral or an extended mineral coating is generatedon the inner pore surface, and/or pluralities of discrete mineralislands.

[0070] Methods for controlling the surface mineralization of biomaterialpolymers comprise altering the molecular weight, polymer composition,ratio of components within the polymer, fabrication technique or surfaceproperties of the biomaterial polymer prior to executing at least afirst surface mineralization process. The methods allow control of thetype of surface mineralization and the degree of surface mineralization,exemplified by the number or size of mineral islands at the surface ofthe biomaterial polymer.

[0071] In one example, the biomaterial polymer is apolylactic-co-glycolic acid copolymer biomaterial and the ratio oflactide and glycolide components within the copolymer composition isaltered. In another example, at least a first surface property of thepolymer composition is altered. Further, controlled surface defects maybe provided to the polymer composition to provide a controllednucleation of discrete mineral islands at the surface of the biomaterialpolymer. The density of such surface defects may be altered.

[0072] The time period of the surface mineralization process may also bealtered. For example, the time of the surface mineralization process maybe extended until discrete mineral islands at the surface of thebiomaterial polymer expand to form a substantially homogeneous mineralcoating at the surface of the biomaterial polymer.

[0073] In all such methods, the mineral-containing solution may be abody fluid or a synthetic medium that mimics a body fluid. Thebiocompatible material may be contacted with the mineral-containingsolution by exposure to a natural or synthetic mineral-containingsolution in vitro or to a mineral-containing body fluid in vivo.

[0074] Any of the foregoing methods, whether for patterning ormineralization or both, are suitable for direct use with, or foradaptation for use with, virtually any biocompatible material or device.For example, the biocompatible materials may comprise at least a firstportion that is biodegradable, non-biodegradable, 3-dimensional,scaffold-like, substantially 2-dimensional, 2-dimensional or film-like.The biocompatible materials may comprise at least a first portion thathas an interconnected or open pore structure,

[0075] The biocompatible materials may further comprise at least a firstportion that is fabricated from metal, bioglass, aluminate, biomineral,bioceramic, titanium, biomineral-coated titanium, hydroxyapatite,carbonated hydroxyapatite, calcium carbonate, or from anaturally-occurring or synthetic polymer portion. The polymers may beselected from collagen, alginate, fibrin, matrigel, modified alginate,elastin, chitosan, gelatin, poly(vinyl alcohol), poly(ethylene glycol),pluronic, poly(vinylpyrollidone), hydroxyethyl cellulose, hydroxypropylcellulose, carboxymethyl cellulose, poly(ethylene terephthalate),poly(anhydride), poly(propylene fumarate), a polymer enriched incarboxylic acid groups, polylactic acid (PLA) polymer, polyglycolic acid(PGA) polymer, polylactic-co-glycolic acid (PLG) copolymer and PLGcopolymers having a ratio of about 85 percent lactide to about 15percent glycolide.

[0076] The biocompatible materials may further comprise at least a firstportion that is prepared by a process comprising gas foaming andparticulate leaching, optionally wherein at least a first bioactivesubstance is operatively associated with the biocompatible materialduring the gas foaming and particulate leaching process.

[0077] The gas foaming and particulate leaching process may comprise thesteps of:

[0078] (a) preparing an admixture at least comprising a leachableparticulate material and particles capable of forming a porous,degradable polymer biomaterial;

[0079] (b) subjecting the admixture to a gas foaming process to create aporous, degradable polymer biomaterial that comprises the leachableparticulate material; and

[0080] (c) subjecting the porous, degradable polymer biomaterial to aleaching process that removes the leachable particulate material fromthe porous, degradable polymer biomaterial, thereby creating additionalporosity.

[0081] The leaching process may comprise contacting the porous,degradable polymer biomaterial with a mineral-containing leachingmaterial.

[0082] The biocompatible materials may further comprise at least a firstportion that is a substantially level surface or a contoured surface. Assuch, the biocompatible material may be fabricated as at least a portionof an implantable device.

[0083] The foregoing methods and resultant biocompatible materials anddevices may further comprise a biologically effective amount of at leasta first bioactive substance, bioactive drug or biological cell, two suchbioactive substances, drugs or biological cells or a plurality of suchbioactive substances, drugs or biological cells.

[0084] Patterned biocompatible materials may thus be exposed to at leasta first binding-competent mineral, bioactive substance or biologicalcell, thereby forming a biocompatible material comprising a mineral,bioactive substance or biological cell bound in a pattern to at least afirst surface thereof. Any resultant patterned mineralized biocompatiblematerials may be exposed to at least a first mineral-adherent cell,thereby forming a mineralized biocompatible material comprising at leasta first cell bound in a pattern to at least a first surface of saidbiocompatible material.

[0085] Growth factors and/or adhesion ligands may be used to forminggrowth factor- or adhesion ligand-coated biocompatible materialscomprising at least a first growth factor or adhesion ligand bound in apattern to at least a first surface of said biocompatible material. Suchgrowth factor- or adhesion ligand-coated biocompatible material may beexposed to at least a first growth factor- or adhesion ligand-adherentcell, thereby forming a mineralized biocompatible material comprising atleast a first cell bound in a pattern to at least a first surface ofsaid biocompatible material.

[0086] The bioactive substance(s) include DNA molecules, RNA molecules,antisense nucleic acids, ribozymes, plasmids, expression vectors, viralvectors, recombinant viruses, marker proteins, transcription orelongation factors, cell cycle control proteins, kinases, phosphatases,DNA repair proteins, oncogenes, tumor suppressors, angiogenic proteins,anti-angiogenic proteins, cell surface receptors, accessory signalingmolecules, transport proteins, enzymes, anti-bacterial agents,anti-viral agents, antigens, immunogens, apoptosis-inducing agents,anti-apoptosis agents and cytotoxins.

[0087] The bioactive substance(s) further include hormones,neurotransmitters, growth factors, hormone, neurotransmitter or growthfactor receptors, interferons, interleukins, chemokines, cytokines,colony stimulating factors, chemotactic factors, extracellular matrixcomponents, and adhesion molecules, ligands and peptides; such as growthhormone, parathyroid hormone (PTH), bone morphogenetic protein (BMP),transforming growth factor-α (TGF-α), TGF-β1, TGF-β2, fibroblast growthfactor (FGF), granulocyte/macrophage colony stimulating factor (GMCSF),epidermal growth factor (EGF), platelet derived growth factor (PDGF),insulin-like growth factor (IGF), scatter factor/hepatocyte growthfactor (HGF), fibrin, collagen, fibronectin, vitronectin, hyaluronicacid, an RGD-containing peptide or polypeptide, an angiopoietin andvascular endothelial cell growth factor (VEGF).

[0088] The biologic cells include bone progenitor cells, fibroblasts,endothelial cells, endothelial cell precursors, stem cells, macrophages,fibroblasts, vascular cells, osteoblasts, chondroblasts, osteoclasts andrecombinant cells that express exogenous nucleic acid segment(s) thatproduce transcriptional or translated products in the cells.

[0089] The biocompatible materials may further comprise a combinedbiologically effective amount of at least a first bioactive substanceand at least a first biological cell. For example, a combinedbiologically effective amount of at least a first osteotropic growthfactor or osteotropic growth factor nucleic acid and a cell populationcomprising bone progenitor cells; or a combined biologically effectiveamount of VEGF or a VEGF nucleic acid and a cell population comprising.

[0090] The at least a first bioactive substance, drug or biological cellmay be incorporated into the biocompatible material prior to, during orsubsequent to the surface-modification process. The incorporation intopatterned surface(s) is an advantage as the bioactive substance, drug orbiological cell is bound in a pattern at the patterned surface. Thebiocompatible material may comprise at least a first mineralizedsurface, wherein a mineral-adherent bioactive substance, drug orbiological cell may be bound to the mineralized surface.

[0091] The present invention further covers all surface-modifiedbiocompatible materials, kits, structures, devices and implantablebiomedical devices with at least a first portion made by any of theforegoing methods, process or means and combinations thereof. Suchsurface-modified biocompatible materials may be used in cell culture,cell transplantation, tissue engineering and/or guided tissueregeneration and in the preparation of one or more medicaments ortherapeutic kits for use for treating a medical condition in need ofcell transplantation, tissue engineering and/or guided tissueregeneration.

[0092] Methods of the invention include those for culturing cells,comprising growing a cell population in contact with a surface-modifiedbiocompatible material in accordance with the present invention. Thecell population may be maintained in contact with the surface-modifiedbiocompatible material under conditions and for a period of timeeffective to generate a two or three dimensional tissue-like structure,such as a bone-like tissue or neovascularized or vascularized tissue.

[0093] Such methods may be executed in vitro or in vivo. The culturedcells may be separated from a surface-modified biocompatible materialand provided to an animal, or may be provided to an animal whilst stillin contact with the surface-modified biocompatible material.

[0094] Further methods include those for transplanting cells into ananimal, comprising applying to a tissue site of an animal a biologicallyeffective amount of a cell-biocompatible material composition thatcomprises a cell population in operative association with asurface-modified biocompatible material in accordance with the presentinvention.

[0095] Still further methods are those for tissue engineering in ananimal, comprising applying to a tissue progenitor site of an animal abiologically effective amount of a biocompatible material compositionthat provides a scaffold for tissue growth and that comprises asurface-modified biocompatible material in accordance with the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0096] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0097]FIG. 1. FTIR spectra displaying development of phosphate (*) andcarbonate ({circumflex over ( )}) peaks with increasing incubation timein SBF. Peaks representing Poly(lactic-co-glycolic acid) are alsolabelled (o). Incubation times are given to the right of each spectrum.

[0098]FIG. 2. Percent mass increase vs. incubation time of scaffoldsincubated in SBF (o), and (▪) control samples incubated in Tris buffer(pH=7.4). Graph shows a trend of increasing mass of SBF-incubatedscaffolds, culminating in a 11+/−2% mass increase after a 16 dayincubation.

[0099]FIG. 3. The mass of phosphate present in the scaffolds vs.incubation time.

[0100]FIG. 4. Compressive modulus vs. incubation time for scaffoldsincubated in SBF (o), and control scaffolds incubated in Tris buffer(pH=7.4) (

).

[0101]FIG. 5. Percent mass increase vs. incubation time for PLGscaffolds incubated in simulated body fluid (SBF). Values represent meanand standard deviation (n=3).

[0102]FIG. 6. The mass of phosphate present in scaffolds vs. incubationtime in SBF. Values represent mean and standard deviation (n=3).

[0103]FIG. 7. Cumulative release of vascular endothelial cell growthfactor (VEGF) from mineralized (X) and non-mineralized [(ν)]▪ scaffolds.Values represent mean and standard deviation (n=5).

[0104]FIG. 8A. Stimulatory effect of VEGF release from mineralized [(ν)▪and non-mineralized [(π)] (□) scaffolds on human derrnal microvascularendothelial cells. Cell counts for each release time interval are givenas percents of the control value (striped column) for that interval.Values that are significantly larger than their corresponding controlare indicated by *'s. Values represent mean and standard deviation(n=5).

[0105]FIG. 8B. Sample dose-response curve demonstrating the mitogeniceffect of VEGF on human dermal microvascular endothelial cells. Valuesrepresent mean and standard deviation (n=5).

[0106]FIG. 9. Incorporation of VEGF into PLG scaffolds during incubationin SBF (♦) and Tris-HCl buffer [(λ)] (o).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0107] Orthopaedic tissue engineering strategies have often focused onthe use of natural or synthetic, degradable materials as scaffolds forcell transplantation (cell-based strategies) (Langer and Vacanti, 1993;Crane et al., 1995; Putnam and Mooney, 1996) or as a means to guideregeneration by native osteogenic cells (conductive strategies) (Minabe,1991). Conductive and cell transplantation strategies have been somewhateffective in bone tissue engineering (Ripamonti, 1992; Ishaug-Riley,1997; Shea et al., “Bone formation from pre-osteoblasts on 3-Dscaffolds,” Submitted). The degree of success of these tissueengineering methods is dependent on the properties of the scaffold.

[0108] Basic scaffold design requirements include degradability,biocompatibility, high surface area/volume ratio, osteoconductivity, andmechanical integrity. A biocompatible scaffold material that isdegradable over a controllable time scale into non-toxic degradationproducts may disappear in concert with new tissue formation, leaving anatural tissue replacement. A high surface area/volume ratio allows formass transport between cells within the scaffold and the surroundinghost tissue, and provides space for ingrowth of fibrovascular tissue.

[0109] Osteoconductivity is important for binding and migration oftransplanted and/or native osteogenic cells. Mechanical integrity isrequired to withstand cellular contractile forces during tissuedevelopment to ensure maintenance of the initial shape of the scaffold(Kim and Mooney, 1998).

[0110] The degradability, biocompatibility, and large surfacearea/volume ratio of scaffolds can be accomplished by the appropriatechoice of synthetic or natural material and processing approach.Poly(lactic acid), poly(glycolic acid), and their copolymers have beenused in tissue engineering applications because they undergocontrollable hydrolytic degradation into natural metabolites (Gilding,1981; Li et al., 1990), and can be processed into highly porousscaffolds by a variety of methods (Harris et al., 1998; Lo et al., 1995;Mikos and Thorsen, 1994).

[0111] A limitation in engineering of many tissue types, including bonetissue, is the inability to induce rapid vascular ingrowth during tissuedevelopment (Mooney et al., 1997). The viability of transplanted cellsand/or host cells that migrate into the scaffold from the native tissueis dependent on transport of nutrients and waste products between thecells and the host tissue.

[0112] Transport is initially due solely to diffusion, and cells morethan several hundred microns from blood vessels in the surroundingtissue either fail to engraft or rapidly die due to oxygen deprivation(Colton, 1995). Studies indicate that blood vessels will infiltrate amacroporous scaffold to provide enhanced transport to engineeredtissues, but the process occurs at a rate of less than 1 mm per day andit typically takes one to two wk for complete penetration of vasculartissue into relatively thin (e.g., 3 mm thick) scaffolds (Mikos et al.,1993; Mooney et al., 1994).

[0113] The present invention provides improved biomaterials for use intissue engineering. Various of the foregoing drawbacks are overcome bythe developments of the invention. Certain materials provided arebiodegradable, porous polymers with homogeneous surface layers ofminerals and mineralized inner pores. Porous polymer materials are alsoprovided that have continuous mineral layers in combination withbioactive factors. Other materials provided are patterned materials, towhich any mineral and/or biological component may be bound in aspatially controlled manner.

[0114] The patterned and/or mineralized polymers with bioactive factorsare provided to give more control over, or to augment, the processes oftissue formation and regeneration. For example, growth factors can beused that induce cells to behave in a specific manner (Giannobile,1996). Several factors have been identified that induce chemotaxis,proliferation, differentiation, and matrix synthesis of specific celltypes, any one or more of which may be used in the present invention.

[0115] Although certain systems have been proposed for factor delivery(Langer, 1990; Whang et al., 1998; Wheeler et al., 1998; Shea et al.,1999; Sheridan et al., J. Cont. Rel., In Press), macroporous tissueengineering matrices are still in need of improvement. The inventorsreasoned that the inclusion of bioactive factors into a scaffold wouldallow a higher level of control over cell function within and adjacentto a scaffold construct, thus addressing specific limitations inconductive and cell-based tissue engineering methods.

[0116] Certain aspects of the present invention therefore providescaffolds that combine the degradability, biocompatibility andosteoconductivity of mineralized scaffolds with the tissue inductiveproperties of bioactive polypeptides. Patterning provides an additionaldegree of control. The invention achieves the growth of bone-likemineral on the inner pore surfaces of a scaffold containing a growthfactor without compromising factor bioactivity or scaffold porosity. Thegrowth factor is exemplified by vascular endothelial cell growth factor(VEGF), a potent mitogen for human micro and macrovascular endothelialcells, which does not exhibit mitogenic effects on other cell types(Leung et al., 1989).

[0117] The mineral- and VEGF-containing matrices of the presentinvention are particularly contemplated for use in inducingneovascularization concurrent with the engineering of bone tissue.Enhanced vascular tissue formation during tissue development will leadto enhanced viability of native and/or transplanted osteogenic cellswithin a scaffold, enabling the engineering of a larger volume of bonetissue.

[0118] Other bioactive factors for use in this invention include growthhormone (GH); parathyroid hormone (PTH, including PTH1-34); bonemorphogenetic proteins (BMPs), such as BMP-2A, BMP-2B, BMP-3, BMP-4,BMP-5, BMP-6, BMP-7 and BMP-8; transforming growth factor-α (TGF-α),TGF-β1 and TGF-β2; fibroblast growth factor (FGF);granulocyte/macrophage colony stimulating factor (GMCSF); epidermalgrowth factor (EGF); platelet derived growth factor (PDGF); aninsulin-like growth factor (IGF) and leukemia inhibitory factor (LIF).

[0119] In fact, virtually any hormone, neurotransmitter, growth factor,growth factor receptor, interferon, interleukin, chemokine, cytokine,colony stimulating factor and/or chemotactic factor protein orpolypeptide may be employed. Further examples include transcription orelongation factors, cell cycle control proteins, kinases, phosphatases,DNA repair proteins, oncogenes, tumor suppressors, angiogenic proteins,anti-angiogenic proteins, immune response stimulating proteins, cellsurface receptors, accessory signaling molecules, transport proteins,enzymes, anti-bacterial and/or anti-viral proteins or polypeptides, andthe like, depending on the intended use of the ultimate composition.

[0120] The biomaterials of the invention with three-dimensionalpatterned surfaces allow location-controlled mineralization and cellulardeposition. The three-dimensional, surface-patterned biomaterials of thepresent invention are “smart biomaterials”, which preferentially bindbiological molecules and cells in specific locations. Theregion-specific surface properties allow control over the location andactivity of cells attaching to the biomaterial, when used both in cellculture and in in vivo implantation.

[0121] These aspects of the invention represent an important advance, ascontrol over the “locations” of cell deposition and activity isenvisioned to be at least as important as controlling the“characteristics” of cell activity. In fact, controlling the location ofactive cells on the surface of a biomaterial may prove to be the mostimportant determinant in tissue regeneration. The techniques of thepresent invention are particularly advantageous as they provide theability to control the locations of cell presence and activity on thesurface of a biomaterial on a micron scale.

[0122] A. Extended Mineral Formation

[0123] Certain aspects of the present invention are processes foraltering a biomaterial by growing an extended or homogeneous minerallayer on the surface. Porous, degradable, polymer biomaterials arepreferred for such processes, e.g., polylactic acid (PLA), polyglycolicacid (PGA) and polylactic-co-glycolic acid (PLGA).

[0124] The inventors' rationale behind coating these materials withminerals is that mineral-like coatings are important for bone growthinto a porous material and/or for adhesion to a substrate. The basis forthis process lies in the observation that in nature, organisms usevarious macromolecules to control nucleation and growth of mineralphases (Campbell et al., 1996; Lowenstein and Weiner, 1989). Thesemacromolecules usually contain functional groups that are negativelycharged at the crystallization pH (Weiner, 1986). It is hypothesizedthat these groups chelate ionic species present in the surroundingmedia, stimulating crystal nucleation (Campbell et al., 1996).

[0125] Observations on natural mineralization processes have notpreviously been adapted for use in connection with biomaterials ortissue engineering processes. However, the present inventors realizedthat a biomaterial substrate could be functionalized in the laboratoryto allow the induction of mineral deposition.

[0126] The inventors further realized that the presence of an extendedor homogeneous mineral layer on the surface of a biomaterial will aid inthe ability to effectively regenerate bone tissue. Described herein arevarious methods for achieving such extended or homogeneous surfacemineralization. However, patterned (or heterogeneous) surfacemineralization is also contemplated for use in certain embodiments, andmay be advantageously achieved by the patterning techniques disclosedherein.

[0127] B. Controlling Locations of Cell Activity

[0128] Recent advances in the control of cellular processes have shownthe utility of controlling the characteristics of cell activity.However, such work has not addressed the specific control of locationsof cell adhesion to a biomaterial. The present inventors envision thecontrol over “locations” of cell activity to be as important as controlof the characteristics of cell activity.

[0129] Specifically, in the area of bone tissue regeneration, aprerequisite for biomaterials to bond to living bone is the formation ofa bone-like mineral layer on the biomaterial in the body. Thisobservation suggested to the inventors that the presence or absence ofthe mineral may determine whether or not bone cells will adhere to andsubsequently act on a biomaterial. Thus, they reasoned that specificcontrol over the location of mineral on a biomaterial surface will allowcontrol over locations of bone cell activity. Patterning of minerals onthe surface of a biomaterial should therefore have a profound effect onthe properties of regenerated bone tissue.

[0130] In order to control the locations of cell adhesion to a threedimensional polymer surface, the present invention further providesseveral new methods of treating biopolymers prior to cell seeding. In asurprisingly simple method, pre-treating only certain regions orsub-sections of biopolymer materials, matrices or scaffolds withmineral-containing aqueous solutions results in localized mineralizationin only those areas in contact with the mineralizing solution. Treatmentof the polymer biomaterials on a micron scale is preferably accomplishedusing one of two different processes: surface photolysis and/or surfaceelectrolysis.

[0131] The patterned photolysis and electrolysis methods of the presentinvention are suitable for use with porous, biodegradable polymerscaffolds. The surface manipulation methods of the present invention aresurprising in that the inventors' adaptation of techniques from theelectron beam lithography field has allowed, for the first time,patterning applications on three dimensional biomatrices, rather thanbeing limited to two dimensions. Three dimensional matrices aregenerally more effective for creating a biomaterial scaffold for use intissue regeneration.

[0132] The various patterning techniques of the invention thereforeprovide the ability to control locations for bone cells specifically(mineral patterning), and for all cell types in general (polymer surfacetreatment without mineral formation). The differences in processing forcontrol of bone cells versus control of other cell types are describedherein.

[0133] As also described in the Detailed Examples, all processes of thepresent invention are room temperature processes. Therefore, specificbioactive substances, drugs and proteins, such as adhesion molecules,cytokines, growth factors and the like, can be incorporated into thepatterning process and resultant biomaterial. Proteins can also beincorporated into the cell culture medium, thus patterning the materialsurface and causing attachment of specific cells.

[0134] C. Photolysis

[0135] Certain methods of the invention for functionalizing the surfacea biomaterial to allow mineralization and/or control of cellularlocation are based upon radiation processes (with or withoutpatterning). To achieve a homogeneous mineral layer on a biomaterialsurface, radiation processes without patterning are used. To achievecontrol of cellular location or mineralization, patterned radiationprocesses are used.

[0136] The treatment of polymer biomaterials with electromagnetic (EM)radiation causes surface degradation via a photolysis reaction. Suitableradiation includes all wavelengths of EM radiation, includingultraviolet, visible, infrared, etc. This form of surface degradation,like that achieved with NaOH, causes an increase in the amount of polaroxygen functional groups on the surface of the material.

[0137] Interpreting results from distinct studies on bone bondingpolymers (Li et al. 1997), the inventors reasoned that the polar oxygengroups formed would spur mineral nucleation on the surface of thebiomaterial when placed in a body fluid. Thus, the inventors realizedthat the ability to pattern three dimensional surface functional groupswould result in the ability to pattern mineral formation and celladhesion on the surface of a biomaterial.

[0138] Unfortunately, the EM radiation techniques formerly availablewere all limited to applications with two dimensional objects.Conventional “contact” optical lithography techniques are so-limited (totwo dimensions) due to the requirement for close contact between a maskor contact grating and the object to be patterned. Thus, prior to thepresent invention, there was no mechanism for producing surface patternson the type of three dimensional, surface-contoured materials that areof most use in tissue engineering.

[0139] Lithographic techniques are based upon passing monochromatic EMradiation through an optical grating to produce radiation patterns on ascreen that is on the opposite side of the grating from the EM radiationsource. The pattern formed can be as simple as equally spaced fringesformed by a grating containing equally spaced slits, or as complicatedas a complex hologram.

[0140] The present invention significantly advances the tissueengineering art by providing methods for using EM radiation to patternthree dimensional biopolymers. In the inventive methods, the “screen” isthe polymer biomaterial. This system amounts to a “diffractionlithography” approach, but the process differs from conventional“contact” optical lithography in that the grating does not act as a maskfor the polymer, so that near contact between the grating and thepolymer is not necessary.

[0141] In the present methods, the grating produces a pattern ofconstructive and destructive interference on the polymer surface. As thegrating is not required to be in near contact with the biomaterialduring treatment, this diffraction lithography process can be used totreat materials with complex three-dimensional surface contours. This isalso a surprising application of previous technology in that thetechnique now employed would sacrifice line width when used in previousembodiments, so, absent the inventors' particular insight regardingthree dimensional matrix patterning, there would be no motivation todevelop this methodology. Further, the “contact mask” does not need tobe removed, improving the sterile nature of the biotechnique.

[0142] Certain types of three dimensional biomatrices envisioned forpatterning using this invention are microsphere and cylindricalmatrices. Although a motivation for developing the present invention wasthe inventors' goal to develop a process for three dimensional andcontoured patterning, now that the process has been developed, it isequally suitable for use with two dimensional polymers.

[0143] D. Electrolysis

[0144] The treatment of polymer biomaterials with electron beam (e-beam)irradiation can also be used to cause surface degradation via anelectrolysis reaction. Surface degradation effects an increase in theamount of polar oxygen functional groups on the surface of the material,which have the same advantageous qualities described herein for thehydrolysis and photolysis reactions.

[0145] Surface electrolysis can be patterned on a polymer surface usinga scanning electron microscope with basic e-beam lithographycapabilities. As shown in the Detailed Examples, this process can alsobe used to treat materials with flat surfaces or complexthree-dimensional surface contours.

[0146] E. Chemical Hydrolysis

[0147] Other methods for surface-functionalizing a biomaterial to allowmineral deposition utilize chemical pre-treatment to achieve surfacehydrolysis, e.g., using a NaOH solution. Surface degradation by thistechnique causes an increase in the amount of polar oxygen functionalgroups on the surface of the material. The functionalized surface isthen incubated in a mineral-containing solution. The inventors have usedsuch functionalization techniques to allow the generation of a mineralcoating or “hypermineralization”.

[0148] Gao et al. (1998) recently reported the surface hydrolysis ofpoly(glycolic acid) meshes in order to increase the seeding density andimprove attachment of vascular smooth muscle cells. Although theirprocedure was also based upon the hydrolysis of PGA in NaOH, the polymerscaffold was then directly progressed to the cell seeding experiments(Gao et al., 1998). The present invention instead exposes thesurface-hydrolyzed biopolymer to a calcium-rich solution to inducesurface mineralization.

[0149] F. Combined Chemical Hydrolysis and Mineralization

[0150] In an unexpected development of the surface-functionalizationmethods, the inventors surprising found that effective mineraldeposition could be achieved on biomaterial surfaces without chemicalpre-treatment. In these methods, a degree of surface hydrolysissufficient to allow mineralization occurs by simply soaking thebiomaterial in an aqueous mineralizing medium. Although pre-treatment,such as by exposure to a NaOH solution, may still be utilized or evenpreferred in certain embodiments, the one-step mineralization processeshave the advantage of simplicity and are preferred in certainembodiments.

[0151] The one-step mineralization methods utilize the same type ofmineral-containing aqueous solutions as described above, such as bodyfluids and synthetic media that mimic body fluids. Functionalization isfollowed by mineralization in situ, without external manipulation.Although these methods are suitable for use with a wide range ofbiopolymers, the current preferences are for use in conjunction with PLGcopolymers with ratios in the region of 85:15 PLG copolymers, and withbiomaterial scaffolds prepared by gas-foaming/particulate leachingprocesses. The use of 85:15 PLG copolymer scaffolds prepared bygas-foaming/particulate leaching is particularly preferred.

[0152] The use of 85:15 PLG copolymers is advantageous as a decrease inthe lactide/glycolide ratio of the copolymer is believed to increase therate of surface hydrolysis. However, prior to the present invention, theuse of 85:15 PLG copolymers was disfavored as the mechanical integrityof the polymer declines with increasing glycolide content. Thisinvention shows that 85:15 PLG copolymers can be used effectively as therapid surface hydrolysis allows sufficient mineral formation to offsetthe potential for decreasing integrity, resulting in a sufficient oreven increased overall strength of the composite.

[0153] The successes of the present one step mineralization methods(Example V), even without foaming/particulate leaching, are in markedcontrast to previous attempts to grow minerals on polyester surfaces.The earlier methods do not result in growth of continuous bone likemineral layers, even after a 6 day incubation in fluids with 50% higherionic concentrations than presently used (Tanahashi et al., 1995).Equally, a 15 day incubation in essentially the same media fluid aspresently used failed to produce continuity of mineral microparticles(Zhang and Ma, 1999).

[0154] The inventors believe that matrix preparation via gasfoaming/particulate leaching techniques results in more surfacecarboxylic acid groups than matrix preparation by other methods (e.g.,solvent casting/particulate leaching). This greater surfacefunctionalization is proposed to contribute to the more rapid nucleationand growth of apatitic mineral observed during the one-stepmineralization processes. Also, the leaching steps of the gasfoaming/particulate leaching methods typically employ mineral solutions,such as 0.1 M CaCl₂, which may further facilitate Ca²⁺ chelation andmore rapid bone-like mineral nucleation.

[0155] The techniques of matrix formation by gas foaming/particulateleaching, with or without additional bioactive agents, are described inthe following co-owned applications, each of which are incorporatedherein by reference without disclaimer: U.S. patent application Ser. No.09/402,119, filed Sep. 20, 1999, which claims priority to PCTApplication No. PCT/US98/06188 (WO 98/44027), filed March31, 1998, whichdesignates the United States and claims priority to U.S. ProvisionalApplication Ser. No. 60/042,198, filed Mar. 31, 1997; and U.S.application Ser. No. 09/310,802, filed May 12, 1999, which claimspriority to second provisional application Ser. No. 60/109,054, filedNov. 19, 1998 and to first provisional application Ser. No. 60/085,305,filed May 13, 1998.

[0156] The studies in Example IX show that bone-like mineral can beadvantageously formed on the inner pore surfaces of matrices prepared bygas foaming/particulate leaching. PLG scaffolds prepared by a gasfoaming/particulate leaching process were successfully mineralized usinga one step incubation in simulated body fluid (SBF) without anyappreciable decrease in total scaffold porosity.

[0157] Using gas foamed/particulate leached PLG scaffolds, a 5 dayincubation in SBF is sufficient for continuous growth of bone-likemineral on the inner pore surfaces of the scaffold (Example IX).Quantification of percent mass gain and phosphate content suggests thatthe majority of mineral growth in these aspects of the invention occursbetween day 2 and day 4 of incubation. These results are even moreadvanced over the previous attempts to produce bone-like minerals onpolyester surfaces (Tanahashi et al., 1995; Zhang and Ma, 1999).

[0158] As these one-step mineralization processes are effective at roomtemperatures, their use to prepare mineralized or hypermineralizedpolymer scaffolds extends to the preparation of mineralized materialsthat include other bioactive substances. It is demonstrated herein thatsuch processes are not detrimental to the activity of biologicalmolecules, such as growth factors. The time- and labor-saving nature ofthese processes therefore make them ideal for preparing matrices for usein many biological processes, especially to stimulate bone growth, whereminerals and growth factors act in concert. The phase, morphology andconstitution of the deposited mineral can be controlled by varying thepH, ionic concentrations and/or temperatures used in the process.

[0159] These mineralization techniques are particularly suitable for usewith biodegradable materials. The ability to obtain a continuousbone-like mineral layer within the pores of a three dimensional, porous,degradable scaffold represents a breakthrough in biomaterial processing.The growth of such a continuous bone-like mineral layer is not onlyimportant to cell seeding, but will also likely increase the mechanicalintegrity of these synthetic constructs via a reinforcement mechanism.

[0160] Polymer constructs used for tissue engineering applications aregenerally highly porous and do not have mechanical properties in thesame range as bone. Creating an interconnected mineral coating over theinner pore surfaces of a polymer construct, according to these aspectsof the present invention, is therefore a distinct advantage. Thesemethods allow for the production of a hard and stiff exoskeleton,increasing the modulus of a biomaterial and enhancing its resistance tocellular contractile forces during tissue development.

[0161] The mineralized scaffold materials of the present invention,e.g., as produced in Example V, in fact have a post-treatmentcompressive modulus larger than those of other poly(α-hydroxy acid)materials used for bone tissue engineering and larger than PLLA bondedpoly(glycolic acid) (PGA) matrices that are adequate for resistance ofcellular forces during smooth muscle tissue development. The materialsof this invention therefore exhibit shape memory, an important factor intissue regeneration. The present invention also provides methods toachieve increases in compressive moduli without notably decreasingscaffold porosity or pore size, another long-sought after designadvantage that allows cellular migration and vascular infiltration.

[0162] G. Mineralization and Growth Factor Release

[0163] In addition to showing successful mineralization using a onestep, five day incubation, the studies of Example IX also demonstratethe sustained release of a bioactive factor (VEGF) from mineralized PLGscaffolds. Three dimensional, porous scaffolds of the copolymer 85:15poly(lactide-co-glycolide) were fabricated by including the growthfactor into a gas foaming/particulate leaching process. The scaffold wasthen mineralized via incubation in a simulated body fluid.

[0164] To summarize, the growth of a bone like mineral film on the innerpore surfaces of the porous scaffold is confirmed by mass increasemeasurements and quantification of phosphate content within scaffolds.Release of ¹²⁵]-labelled VEGF was tracked over a 15 day period todetermine release kinetics from the mineralized scaffolds. Sustainedrelease from the mineralized scaffolds was achieved, and growth of themineral film altered the release kinetics from the scaffolds byattenuating the initial burst effect, and making the release curve morelinear. The VEGF released from the mineralized and non-mineralizedscaffolds was over 70% active for up to 12 days following mineralizationtreatment, and the growth of mineral had little effect on total scaffoldporosity.

[0165] In more detail, the mineral presence is shown to slow the releaseof the growth factor from the scaffold, resulting in release of agreater amount of factor for a longer time period (e.g., days 3 through10). After an initial burst release of 44±2% of the incorporated factorin the first 36 h, the release profile is sustained from the mineralizedsponges for up to 10 days in SBF (FIG. 7). In contrast, the release fromthe non-mineralized scaffolds shows a relatively large initial burst of64±2% over the first 60 h, followed by sustained release for ˜5 days.

[0166] The release of a bioactive factor from a mineralized scaffold isan important result for tissue engineering, particularly bone tissueengineering, because it combines the osteoconductive qualities of abone-like mineral with the tissue inductive qualities of a bioactivefactor, such as a protein growth factor. VEGF release is specificallyuseful in the induction of vascular tissue ingrowth for tissueengineering. This system could also be used with a variety of otherinductive protein growth factors, easily matched to the cell and tissuetypes intended to be stimulated.

[0167] Example IX suggests that the growth of mineral on the surface ofporous PLG modulates factor release. There is a clear correlationbetween the onset of mineral growth and the divergence in the releaseprofiles for samples incubated in SBF and PBS (control). The formeroccurs between day 2 and day 4 of incubation (FIG. 6), while the latteroccurs at the 3 day time point (FIG. 7). The net effect of mineralpresence is attenuation of the initial burst release from the scaffold,and sustained release of a larger amount of factor for a longer periodof time.

[0168] The release modulation effect is also apparent in the bioactivitydata (FIG. 8A). Release from the mineralized scaffolds has asignificantly greater effect on cell proliferation than release from thenon-mineralized scaffolds during the factor release interval 8-10 days.Examination of the release profiles (FIG. 7) indicates that themineralized scaffolds release a larger amount of VEGF than thenon-mineralized scaffolds during this period.

[0169] Previous controlled release formulations using poly(α-hydroxyacid) materials frequently demonstrate a sizeable initial burst in thefirst 1-5 days of release followed by minimal release at later timepoints (Cohen et al., 1991; Kwong et al., 1986). Achieving relativelyconstant release over a longer period of time is a substantial goal inpolymeric drug delivery. Previous attempts to address the “burst effect”have used double-walled polymer microspheres (Pekarek et al., 1994) andmicrospheres encapsulated in microporous membranes (Kreitz et al.,1997). The bone-like mineral in this study achieves a functional effectsimilar to the outer layer in double-walled polymeric drug deliverysystems.

[0170] The formation of a mineral layer within the pores of PLGscaffolds does not notably impair the ability of released growth factorto stimulate proliferation of human dermal microvascular endothelialcells. The possibility of protein denaturation and aggregation uponexposure to moisture is a concern in the controlled release of proteinsfrom certain polymer systems (Ishaug-Riley et al., 1998). In this case,the protein is clearly bioactive for eleven days after mineralizationtreatment (16 days after sample preparation).

[0171] The 11 day time scale was chosen for analysis in this studybecause a large percentage of transplanted cells fail to engraft and diewithin this time period without the development of a vascular supply toaugment mass transport (Mooney et al., 1997; Ishaug-Riley et al., 1998).Sustained release over this time scale induces increased proliferationof endothelial cells, thus supporting angiogenesis during the initialstages of osseous tissue development in vivo.

[0172] The present invention therefore provides a system for thesustained release of bioactive factors, such as polypeptides, growthfactors and hormones, from mineralized PLG scaffolds. Themineral-biofactor-scaffolds have particular uses in orthopaedic tissueengineering. The presence of a bone-like mineral is a prerequisite toconduction of osteogenic cells into various porous synthetic constructs(Hench, 1991; Kokubo, 1991), and so the mineral is associated withincreased bioreactivity (LeGeros and Daculzi, 1990). The mineral grownby the methods of the present invention thus provides enhancedosteoconductivity in addition to the inductive (e.g., angiogenic) effectof protein release. The growth of the mineral is accomplished via asurprisingly simple single step, room temperature process which,importantly, does not compromise growth factor bioactivity, or totalscaffold porosity.

[0173] The following examples are included to demonstrate certainpreferred embodiments of the invention. It will be appreciated by thoseof skill in the art that the compositions and techniques disclosed inthe examples that follow represent compositions and techniquesdiscovered by the inventor to function well in the practice of theinvention, and thus can be considered to constitute certain preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE I Homogeneous Surface Mineralization

[0174] A porous, degradable polymer biomaterial is treated forsubstantially homogeneous mineralization by either pre-treating toinduce surface hydrolysis and then exposing to a mineralizing solution(Examples II through IV) or by conducting a one-step surface hydrolysisand mineralization process (Example V).

[0175] Pre-treatment to produce homogeneous surface hydrolysis may beachieved by either soaking in a NaOH solution (Example II) or bytreating with electromagnetic (EM) radiation (Example III). The treatedbiomaterial is incubated in a mineral-rich, preferably a calcium-rich,fluid, such as a body fluid or synthetic media that mimics body fluid,to spur nucleation and growth of a homogeneous mineral film on thesurface (Example IV).

[0176] Functionalization and concomitant mineralization can also beachieved by simply soaking in mineral-containing aqueous solutions,preferably in body fluids or synthetic media that mimic body fluids.Preparation of the polymer biomaterials using a gas-foaming/particulateleaching process is generally preferred for such one step mineralization(Example V).

[0177] Once mineralized, osteogenic cell precursors are seeded onto thebiomaterial in vitro in a cell culture medium. In vivo, bone cellsattach to the biomaterial when implanted.

EXAMPLE II NaOH Pre-Treatment for Surface Mineralized Films

[0178] PLGA films (˜25 μm thickness) were prepared by a pressure castingtechnique. Raw polymer pellets were loaded into a mold and placed in aconvection oven at 200 degrees C. The molds were heated under pressure(˜22 N) for 30 sec. and then cooled to room temperature.

[0179] For the creation of surface functional groups by NaOH treatment,the films were cleansed and immersed in 1.0 N NaOH solution for varyingtimes, up to 10 minutes to create surface functional groups. Followingimmersion, samples were rinsed 3×in distilled water.

EXAMPLE III UV Pre-Treatment for Surface Mineralized Films

[0180] PLGA films (˜25 μm thickness) were prepared by a pressure castingtechnique. Raw polymer pellets were loaded into a mold and placed in aconvection oven at 200 degrees C. The molds were heated under pressure(˜22 N) for 30 sec. and then cooled to room temperature.

[0181] For the creation of surface functional groups by UV (ultraviolet) treatment, membranes were exposed to up to 8 hrs of surfaceirradiation.

EXAMPLE IV Surface Mineralization after Pre-Treatment

[0182] Membranes treated by either NaOH treatment or UV treatment weresubsequently incubated at 37 degrees C. in 50 ml of a simulatedphysiological fluid (SPF, Na: 142 mM, K: 5 mM, Ca: 2.5 mM, Mg: 1.5 mM,Cl: 148 mM, HCO3: 4.2 mM, HPO4: 1 mM, SO4: 0.5 mm) buffered to pH 7.4.Solutions were replaced every 48 hours to ensure that there weresufficient ions in solution to induce mineral nucleation and growth.Following immersion for periods of 120 to 240 hours, samples were dried.

[0183] Fourier transform infrared (FTIR) analysis indicates the presenceof a surface amorphous apatite. FTIR spectra of scaffolds treated for 0,2, 6, 10, and 16 days indicate the growth of a carbonated apatitemineral within the scaffold (FIG. 1). Equivalent spectra were alsoproduced with the UV-treated films. The broad band at 3570 cm⁻¹ isindicative of the stretching vibration of hydroxyl ions in absorbedwater. The peak at 1454 cm⁻¹ is indicative of CO₃ ²⁻ν₃, while the 867cm⁻¹ represents CO₃ ²⁻ν₂. The peaks at 1097 cm⁻¹ and 555 cm⁻¹ areindicative of anti-symmetric stretch (ν₃) and anti-symmetric bending(ν₄) of PO₄ ³—, respectively. The peak at 1382 cm⁻¹ represents a NO₃band.

[0184] The presence of OH—, CO₃ ²⁻ and PO₄ ³— all indicate that anapatitic layer has been formed. Other bands representative of apatitesare masked because of the strong absorption of the PLGA.

[0185] The major peaks at 1755 cm⁻¹ and 1423 cm⁻¹ represent PLGA, andthe peak at 1134 cm⁻¹ is indicative of C—O stretch in the ester. Thepeaks at 756 cm⁻¹ and 956 cm⁻¹ are indicative of the amorphous domainsof the polymer.

[0186] The scaffolds demonstrated an increase in mass over time,culminating in a 11±2% mass gain at the end of the 16 day incubation(FIG. 2). ANOVA of percent mass changes of experimental scaffolds reveala significant difference in scaffold mass over time (p<0.05), whileANOVA of percent mass changes of control scaffolds does not show asignificant difference over time (p>0.05). Percent mass changes ofexperimental samples and control samples were significantly differentfor each time point beyond 8 days (p<0.05).

[0187] To confirm that the increase in mass was caused by deposition ofan apatitic mineral, the mass of phosphate in the scaffolds was nextanalyzed. Phosphate content within the treated scaffolds also increasedsignificantly with the treatment time (FIG. 3). Comparison of phosphatemasses via ANOVA show a statistically significant increase over time(p<0.05), and the differences in phosphate mass between day 8 and 12(p<0.05) and between day 12 and 14 (p=0.05) were also statisticallysignificant. After a 14 day incubation, estimation of the mass ofmineral on the scaffold using phosphate mass data gives 0.76 mg ofhydroxyapatite, while the measured mass increase of the scaffold is1.02±0.40 mg. The fact that the measured value is larger than theestimated value suggests significant carbonate substitution in themineral crystal.

[0188] Growth of the BLM layer significantly increased the compressivemodulus of 85:15 PLG scaffolds (FIG. 4) without a significant decreasein scaffold porosity. The compressive modulus increased from 60±20 KPabefore treatment to 320±60 KPa after a 16 day treatment, a 5-foldincrease in modulus. ANOVA of modulus changes of experimental scaffoldsreveal a significant difference in scaffold modulus over time (p<0.05),while ANOVA of control modulus data does not show a significantdifference over time (p>0.05). The differences between moduli ofexperimental scaffolds and moduli of control scaffolds werestatistically significant for treatment times of 10 days or longer(p<0.05). The porosity of the scaffolds did not decrease appreciablyafter incubation in SBF. Untreated scaffolds were 95.6±0.2% porous,while scaffolds incubated in SBF for 16 days were 94.0±0.30% porous(n=3). This agrees with the electron micrographs, which displayed only athin (1-10 μm) mineral coating, and thus no significant change in poresize due to mineral growth.

[0189] This example shows the successful use of this room temperatureprocess to yield an apatitic surface layer upon a treated polymersurface. The importance of room temperature processing is thatattachment of biological factors is readily achievable, without concernfor denaturation.

EXAMPLE V One Step Mineralization

[0190] One step, room temperature incubation processes can also be usedto cause nucleation and growth of mineral layers on polymer surfaces.This is achieved by incubating polymer scaffolds in mineral-containingaqueous solutions, such as body fluids and synthetic media that mimicbody fluids. These processes are able to grow bone-like minerals withinpolymer scaffolds in surprisingly simple and inexpensive methods. Theeffectiveness of these methods under room temperature conditions rendersthem conducive to the inclusion of bioactive proteins and othermaterials into the processing mineralization.

[0191] A first example of one step mineralization concerns the mineraldeposition on porous poly(lactide-co-glycolide) sponges via incubationin a simulated body fluid. The simple incubation technique was used toobtain nucleation and growth of a continuous carbonated apatite mineralon the interior pore surfaces of a porous, degradable polymer scaffold.

[0192] A 3-dimensional, porous scaffold of 85:15 PLG was fabricated by asolvent casting/particulate leaching process and incubated in simulatedbody fluid (SBF; NaCl-141 mM, KCl-4.0 mM, MgSO₄-0.0.5 mM, MgCl₂-1.0 mM,NaHCO₃4.2 mM, CaCl₂-2.5 mM, and KH₂PO₄-1.0 mM in deionized H₂O, bufferedto pH=7.4 with Trisma-HCl). Fourier transform IR spectroscopy and SEManalyses after different incubation times demonstrated the growth of acontinuous bone-like apatite layer within pores of the polymer scaffold.

[0193] The majority of the mineral growth occurred between days 8 and12. Mineral growth into a continuous layer likely occurs from day 12,and is complete at or before day 16. The mineral grown, beingcontinuous, is thus similar to that in bones and teeth.

[0194] The scaffolds demonstrated an increase in mass over time, with an11±2% gain after 16 days. The increase in mass is due to deposition ofan apatitic material. Quantification of phosphate on the scaffoldrevealed the growth and development of the mineral film over time withan incorporation of 0.43 mg of phosphate (equivalent to 0.76 mg ofhydroxyapatite) per scaffold after 14 days in SBF. The measured overallmass increase of the scaffold was 1.02±0.4 mg at 14 days. This suggestscarbonate substitution in the mineral crystal.

[0195] The compressive moduli of polymer scaffolds also increasedfivefold with formation of a mineral film after a 16 day incubationtime, as opposed to control scaffolds. This was achieved without asignificant decrease in scaffold porosity. The thin mineral coating isthus functionally important, yet mineralization does not change the poresize.

[0196] As shown in the mineralization and growth factor studies ofExample IX, 85:15 PLG scaffolds prepared by gas foaming/particulateleaching exhibit even more rapid nucleation and growth of apatiticmineral. The 85:15 PLG scaffolds prepared via solventcasting/particulate leaching showed a 3±1% increase in mass after a 6day incubation in SBF. In comparison, 85:15 PLG scaffolds prepared bygas foaming/particulate leaching showed a mass increase of 6±1% after a4 day incubation in SBF.

[0197] The even more rapid nucleation and growth of apatitic mineral on85:15 PLG scaffolds prepared by gas foaming/particulate leaching isbelieved to be due to the increase in carboxylic acid groups caused bythe gas foaming/particulate leaching process, i.e., the greater surfacefunctionalization. Leaching with 0.1 M CaCl₂ also likely facilitateschelation of Ca²⁺ ions, producing more rapid bone-like mineralnucleation.

EXAMPLE VI Bone Cell Control

[0198] Polymer biomaterial is treated to form a patterned biosurface,preferably suing either patterned EM radiation or electron beamirradiation. Treated biomaterial is washed with distilled water toremove residual monomers from the surface photolysis or electrolysis.

[0199] The treated biomaterial is incubated in a mineral-rich,preferably a calcium-rich, fluid, such as a body fluid or syntheticmedia that mimics body fluid, to spur nucleation and growth of mineralon the treated regions of the polymer. This results in a mineral patternon the surface of the polymer. This step can be done either in vitro,using a body fluid or simulated body fluid; or in vivo, where thenatural body fluid performs this function.

[0200] Osteogenic cell precursors are seeded onto the biomaterial invitro in a cell culture medium. In vivo, bone cells attach to thebiomaterial when implanted. In either case, cells adhere preferentiallyto mineralized portions of the substrate.

EXAMPLE VII Diffraction Lithography

[0201] Previous studies on the control of locations of cell adhesion toa biomaterial surface have utilized conventional UV lithography topattern a two dimensional polymer surface (Pierschbacher & Ruoslahti,1984); Ruoslahti & Pierschbacher, 1987); Matsuda et al., 1990); Britlandet al., 1992); Dulcey et al., 1991); Lom et al., 1993); Lopez et al.,1993); Healy et al., 1996).

[0202] In the prior techniques, the two-dimensional biomaterial surfaceis coated with a thin layer of photoresist (PR), the PR is exposedthrough a metal mask, and the exposed PR is removed in solvent, leavinga PR mask on the surface of the biomaterial sample. The surface of thepolymer biomaterial is then chemically or physically treated through thePR mask, and the mask is removed by a solvent after treatment.

[0203] The former processes requires a flat, two dimensionalbiomaterial, which suffices for studying the effects of surfacetreatment on cell activity, but is not sufficient for the treatment oftypical biomaterials, which have three dimensional surface contours.

[0204] In the present methods, suitable for use with three dimensionalpolymers, the grating produces a pattern of constructive and destructiveinterference on the polymer surface. As the grating is not required tobe in near contact with the biomaterial during treatment, thisdiffraction lithography process can be used to treat materials withcomplex three-dimensional surface contours. However, the process isequally useful in connection with two dimensional biomaterials.

EXAMPLE VIII Control of Other Cell Types

[0205] Polymer biomaterial is treated to form a patterned biosurface,preferably using either patterned EM radiation or electron beamirradiation. Treated biomaterial is washed with distilled water toremove residual monomers from the surface photolysis or electrolysis.

[0206] The treated biomaterial is incubated in a solution containingbioactive molecules or proteins, such as growth factors, adhesionmolecules, cytokines and such like, which promote adhesion of a specificcell type. Cells are seeded onto the biomaterial in vitro in a cellculture medium. In vivo, cells attach to the biomaterial when implanted.In either case, cells adhere preferentially to the treated portions ofthe substrate.

[0207] The use of specific agents or proteins, such as growth factors,that promote attachment of certain cell types, gives the potential topattern any cell type on the three dimensional surface of the polymer,both in vitro and in vivo.

EXAMPLE IX Growth Factor Release from Mineralized Matrices

[0208] A. Materials and Methods

[0209] 1. Gas Foaming-Particulate Leaching

[0210] Poly(lactide-co-glycolide) pellets with a lactide:glycolide ratioof 85:15 were obtained from Medisorb, Inc. (I.V.=0.78 dl/g) and groundto a particle size between 106 and 250 μm. Ground PLG particles werethen combined with 250 μl of a 1% alginate (MVM, ProNova; Oslo, Norway)solution in ddH₂O, and 3 μg of vascular endothelial cell growth factor(VEGF, Intergen; Purchase, N.Y.), and vortexed. These solutions werelyophilized, mixed with 100 mg of NaCl particles (250 μm<d<425 μm), andcompression molded at 1500 psi for 1 min in a 4.2 mm diameter die. Thisyielded 2.8 mm thick disks with a diameter of 4.2 mm.

[0211] Disks were then exposed to 850 psi CO₂ gas in an isolatedpressure vessel and allowed to equilibrate for 20 h. The pressure wasdecreased to ambient in 2 min, causing thermodynamic instability, andsubsequent formation of gas pores in the polymer particles. The polymerparticles expand and conglomerate to form a continuous scaffold withentrapped alginate, VEGF, and NaCl particles. After gas foaming, thedisks were incubated in 0.1 M CaCl₂ for 24 h to leach out the saltparticles and induce gellation of the alginate within the polymermatrix. Alginate was included in the scaffolds because it has been shownto abate the release of VEGF from PLG scaffolds (Wheeler et al., 1998).

[0212] 2. Mineralization

[0213] Certain scaffolds were mineralized via a 5 day incubation in asimulated body fluid (SBF). Simulated body fluid (SBF) was prepared bydissolving the following reagents in deionized H₂O: NaCl-141 mM, KCl-4.0mM, MgSO₄0.0.5 mM, MgCl₂-1.0 mM, NaHCO₃0.4.2 mM, CaCl₂-2.5 mM, andKH₂PO₄-1.0 mM. The resulting SBF was buffered to pH=7.4 with Trisma-HCland held at 37° C. during the incubation periods. The SBF solutions wererefreshed daily to ensure adequate ionic concentrations for mineralgrowth.

[0214] The porosity of scaffolds was calculated before and aftermineralization treatment using the known density of the solid polymer,the known density of carbonated apatite, the measured mass of mineraland polymer in the scaffolds, and the volume of the scaffold.

[0215] 3. Characterization of Mineral Growth

[0216] To analyze mineral growth on gas foamed PLG scaffolds, sets ofthree scaffolds were incubated in SBF for periods ranging from 0-10days. Samples were removed from solution and analyzed after 0, 2, 4, 8,and 10 day incubation periods. The dry mass of each scaffold wasmeasured before and after incubation in SBF, and percent increases inmass were calculated and compared using ANOVA and a Student's t-test toreveal significant differences in mass for different SBF incubationtimes.

[0217] The amount of phosphate present in the scaffolds after theaforementioned incubation times was determined using a previouslydescribed colorimetric assay (Murphy et al., J. Biomed. Mat. Res., InPress; incorporated herein by reference). The phosphate mass data werealso compared using ANOVA and a Student's t-test to reveal significantdifferences in mass for different SBF incubation times.

[0218] To estimate the amount of apatite on the scaffold after a 6 dayincubation, the measured mass of phosphate was multiplied by the knownratio of mass of hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂, f.w.=1004.36 g] tomass of phosphate in hydroxyapatite (569.58 g). This is a conservativeestimate, since it assumes that all phosphate is being incorporated intostoichiometric hydroxyapatite. This mineral mass estimate increases ifone assumes increasing substitution of carbonate into the mineralcrystal.

[0219] 4. VEGF Release Measurements

[0220] In order to assess the incorporation efficiency of VEGF into thePLG scaffolds and to track the VEGF release kinetics from the scaffolds,receptor-grade ¹²⁵I-labeled human VEGF (90 μCi/μg; BiomedicalTechnologies Inc.; Stoughton, Mass.) was utilized as a tracer. In placeof the 3 μg VEGF in the normal sample preparation, 0.5 μCi ofradiolabeled VEGF was added to each matrix. To assess VEGF incorporationefficiency, the total incorporated activity was compared to the activityof the initial ¹²⁵I VEGF sample prior to incorporation into thescaffolds.

[0221] To determine the effects of mineral growth on factor release,release kinetics were measured both in SBF during mineral formation andin phosphate buffered saline (PBS). Scaffolds prepared with radiolabeledVEGF were placed in 4 ml of SBF or PBS and held at 37° C. At various settimes, the scaffolds were removed from solution and their radioactivitywas assessed using a gamma counter. After each analysis, solutions wererefreshed and scaffolds were placed back into solution.

[0222] The amount of radiolabeled VEGF released from the scaffolds wasdetermined at each time point by comparing the remaining ¹²⁵I VEGF tothe total originally loaded into each scaffold. The percent release ofVEGF from scaffolds incubated in SBF was compared to that of scaffoldsincubated in PBS at each time point via a Student's t-test to revealsignificant differences in cumulative release.

[0223] 5. Biological Activity of Released VEGF

[0224] The biological activity of VEGF incorporated into, and releasedfrom, polymer matrices was determined by testing its ability tostimulate the growth of cultured human dermal microvascular endothelialcells isolated from neonatal dermis (HMVEC(nd), Cascade Biologics;Portland, Oreg.).

[0225] HMVEC(nd) were cultured to passage 7 in MCDB 131 media (CascadeBiologics) supplemented with Cascade Biologics' microvascular growthsupplement (5% fetal bovine serum, hydrocortisone, human fibroblastgrowth factor, heparin, human epidermal growth factor, and dibutyrylcyclic AMP) prior to use. Cells were plated at a density of 5×10³cells/cm² on 12 well tissue culture dishes (Coming; Cambridge, Mass.)which were precoated with 1 μg/cm² human plasma fibronectin (LifeTechnologies, Grand Island, N.Y.). The cells were allowed to attach for24 h, and the media in each well was replaced then with 3 ml ofserum-free media (Cell Systems; Kirkland, Wash.) supplemented with 50μg/ml gentamicin (Life Technologies).

[0226] A 12 mm transwell (3 μm pore diameter, Coming) containing eithermineralized, or non-mineralized, VEGF releasing matrix was placed ineach experimental well (n=5 for each group), while mineralized matricescontaining no VEGF were placed in the control wells (n=5). To determinethe dose response to known concentrations of VEGF, additional wells (n=4per concentration) were supplemented with 40, 20, 10, and 5 ng/ml ofsoluble VEGF which had not been incorporated into matrices.

[0227] After 72 h all of the cells in the experimental and control wellswere removed with a solution of 0.05% trypsin/0.53 mM EDTA (LifeTechnologies), and counted using a ZM Coulter counter (Coulter; Miami,Fla.). The transwells containing the matrices were immediatelytransferred to new fibronectin-coated (1 μg/cm²) wells that had beenseeded with cells (5×10³ cells/cm²) 24 h before, and allowed to incubatefor an additional 72 h before the cells were removed and counted. A newset of VEGF dose response wells were also set up concurrent with thetransfer of the transwells. The 72 h cycles were continued for 12 days.

[0228] Cell counts in experimental wells were compared to cell counts incontrol wells for each 72 h interval using a Student's t-test to revealsignificant differences in HMVEC proliferation.

[0229] B. Results

[0230] 1. Mineralization

[0231] Incubation of gas foamed 85:15 poly(lactide-co-glycolide)scaffolds containing VEGF resulted in the growth of bone-like mineral onthe inner pore surfaces. Analysis of variance showed that differences inpercent mass gain with SBF incubation time were significant (p<0.05).The scaffolds showed an increase in mass with incubation time, with a6±1% mass gain after a 4 day incubation in SBF (FIG. 5). The scaffoldmass subsequently remained relatively constant. The increase in massbetween two day and four day incubation times was significant (p<0.05),while there was no significant difference in percent mass gain betweenthe four day incubation time and the longer incubation times (p>0.05).

[0232] To verify that the increase in mass was caused by the depositionof an apatitic mineral, the mass of phosphate in the scaffolds wasanalyzed. Phosphate content within scaffolds increased with SBFincubation time (FIG. 6). Analysis of variance showed that differencesin phosphate content with SBF incubation time were significant (p<0.05).The difference in phosphate content between the two day and six dayincubation times was significant (p<0.05), while there was nosignificant difference between the phosphate mass of the six dayincubation time and longer incubation times (p>0.05).

[0233] The inventors have previously shown that the increase in mass andphosphate content in these scaffolds indicates growth of a continuousbone-like mineral film on the inner pore surfaces (Murphy et al., J.Biomed. Mat. Res., In Press).

[0234] The total porosity of the scaffolds after a 10 day incubation inSBF was 92±1%, which is similar to the initial scaffold porosity(93±1%).

[0235] After a 6 day incubation, estimation of the mass of mineral onthe scaffold using phosphate mass data gives 0.10 mg of hydroxyapatite,while the measured mass increase of the scaffold is 0.39±0.03 mg. Thefact that the measured value is larger than the estimated value suggestssignificant carbonate substitution in the mineral crystal.

[0236] 2. VEGF Release and Activity

[0237] Vascular endothelial cell growth factor (VEGF) was incorporatedinto PLG scaffolds with an efficiency of 44±9% and released over a 15day period in SBF and PBS solutions. An initial burst release of theincorporated growth factor was observed over the first 12-36 h followedby a sustained release for the remainder of the study (FIG. 7).

[0238] The cumulative release from scaffolds incubated in SBF becamesignificantly smaller than release from scaffolds incubated in PBS after3 days, and this difference remained significant through 10 days ofrelease (p<0.05). At time points beyond 10 days there is no significantdifference in cumulative release from scaffolds incubated in SBF versusthose incubated in PBS (p>0.05).

[0239] VEGF released from mineralized and non-mineralized scaffolds hada mitogenic effect on human dermal microvascular endothelial cells(HMVECs).

[0240] Cells were grown in wells containing three different scaffoldtypes: 1) Mineralized, VEGF-containing scaffolds (MV scaffolds); 2)non-mineralized, VEGF containing scaffolds (NV scaffolds); and 3)mineralized control scaffolds without VEGF (MC scaffolds). Cells grownin wells containing MV and NV scaffolds demonstrated significantlyincreased proliferation when compared with cells grown in wellscontaining MC scaffolds (FIG. 8A). Cell counts were significantly higherin wells containing MV and NV scaffolds for all time intervals (p<0.05)with the exception of the wells containing NV scaffolds over the 14-16day factor release interval.

[0241] During the 8-10 day factor release interval, MV scaffolds showeda significantly greater mitogenic effect on HMVECs than NV scaffolds(p<0.05). There was no significant difference in the stimulatory effectof MV scaffolds versus NV scaffolds for any other time interval(p>0.05).

[0242] A dose-response curve (FIG. 8B) generated for the HMVECs was usedto calculate an effective concentration for the released growth factor.Comparison of this effective concentration with the amount of VEGF knownto be released during each time interval (FIG. 7) indicates that thereleased VEGF is over 70% active for all time intervals.

EXAMPLE X Effects of Growth Factors on Mineralization

[0243] A. Materials and Methods

[0244] Poly(lactide-co-glycolide) pellets with a lactide:glycolide ratioof 85:15 were obtained from Medisorb, Inc. (I.V.=0.78 dl/g) and groundto a particle size between 106 and 250 μm. Ground PLG particles werethen combined with 250 μl of a 1% alginate (MVM, ProNova; Oslo, Norway)solution in ddH₂O, and vortexed. These solutions were lyophilized, mixedwith 100 mg of NaCl particles (250 μm<d<425 μm), and compression moldedat 1500 psi for 1 minute in a 4.2 mm diameter die. This yielded 2.8 mmthick disks with a diameter of 5.0 mm.

[0245] Disks were then exposed to 850 psi CO₂ gas in an isolatedpressure vessel and allowed to equilibrate for 20 hours. The pressurewas decreased to ambient in 2 minutes, causing thermodynamicinstability, and subsequent formation of gas pores in the polymerparticles. The polymer particles expand and conglomerate to form acontinuous scaffold with entrapped alginate, and NaCl particles. Aftergas foaming, the disks were incubated in 0.1M CaCl₂ for 24 hours toleach out the salt particles and induce gellation of the alginate withinthe polymer matrix. Alginate was included in the scaffolds because ithas been used in VEGF release studies to help abate the release of VEGFfrom PLG [scaffolds¹¹] scaffolds, and it was necessary to preciselymimic the scaffold conditions during factor release studies.

[0246] The total porosity of scaffolds was calculated using the knowndensity of the solid polymer, the measured mass polymer in the scaffold,and the measured volume of the scaffold. Cross sectional electronmicrographs of scaffolds were obtained by bisecting the scaffolds viafreeze fracture and imaging using a Hitachi S3200N scanning electronmicroscope.

[0247] To assess the effect of VEGF in solution on the mineral growthprocess, scaffolds were incubated in SBF containing 0.2 μCi ¹²⁵I VEGF(Receptor grade human VEGF, 90 μCi/μg, Biomedical Technologies Inc.;Stoughton, Mass.) (n=5). Samples were incubated for five days, sincethis is the time period required for growth of a significant amount ofbone-like mineral within the inner pore surfaces of gasfoamed/particulate leached 85:15 PLG [scaffolds¹⁴] scaffolds. Afterincubation, scaffolds were washed three times in [ddH₂O] ddH₂O, andassessed for radioactivity using a

[0248] gamma counter. The percent incorporation of VEGF into the polymerscaffolds was calculated (Counts of scaffold/counts of solution *100)and plotted vs. incubation time.

[0249] The incubation was done in tubes that were siliconized usingsigmacote, then presoaked in a 1% bovine serum albumin (BSA) solutionfor 30 minutes to coat the tube surface with BSA and thus reduce bindingof VEGF to the inner surface of the tubes. Solutions were refresheddaily to ensure sufficient ionic concentrations for mineral growth andconstant concentration of the iodinated growth factor in the solution.

[0250] Simulated body fluid (SBF) was prepared daily by dissolving thefollowing reagents in deionized H₂O: NaCl-141 mM, KCl-4.0 mM, MgSO₄0.0.5mM, MgCl₂-1.0 mM, NaHCO₃— 4.2 mM, CaCl₂-2.5 mM, and KH₂PO₄-1.0 mM. Theresulting SBF was buffered to pH=7.4 with Trisma-HCl and held at 37° C.during the incubation periods.

[0251] B. Results

[0252] 85:15 Poly(lactide-co-glycolide) scaffolds prepared via a gasfoaming/particulate leaching process were 93±1% porous and displayed anopen pore structure with a pore diameter of ˜200 μm.

[0253] The incorporation of radioactive VEGF into the scaffolds waslarger for control scaffolds than for experimental scaffolds for alltime points beyond 2 days (p<0.05). The control data also show a trendof increasing incorporation of VEGF with increasing incubation time(FIG. 9). These data indicate that VEGF is being incorporated into thecontrol scaffolds more efficiently than it is being incorporated intothe experimental samples, and the amount of VEGF in the experimentalscaffolds is not increasing during mineralization treatment.

[0254] The data show that VEGF does not significantly incorporate intoPLG scaffolds during incubation in SBF. There is also no significantincorporation of the growth factor into the mineral during the initialstages of mineral growth. Thus, the previously shown attenuation of VEGFrelease from PLG scaffolds during mineral growth cannot be explained byincorporation of protein into the mineral film, binding of the proteinto the scaffold surface, or diffusion of the protein back into thescaffold during protein release.

[0255] The postulated steps in the mineral growth process on PLGscaffolds are: I) surface functionalization via a hydrolysis reaction;2) Chelation of [Ca2+] Ca²⁺ ions by surface carboxylate anions; 3)Nucleation and growth of mineral crystals on the polymer surface. Thelack of incorporation of VEGF into PLG scaffolds incubated in SBFindicates that the protein does not compete with calcium ions forbinding sites on the inner pore surfaces of the scaffolds or efficientlydiffuse back into the scaffolds after release.

[0256] In this case, the amount of protein incorporated into thescaffolds was significantly larger for control samples incubated inTris-HCl buffer, and the incorporation increased over time. Theincreased efficiency of incorporation of VEGF into control samples maybe due to more efficient diffusion of the factor into control scaffolds,or enhanced binding of the factor to the inner pore surfaces of thecontrol scaffolds. This result shows that the effects of mineral growthon VEGF release from PLG scaffolds cannot be explained incorporation ofVEGF back into PLG scaffolds after release, or binding of VEGF to thescaffold's inner pore surfaces.

[0257] There is no significant incorporation of protein into the mineralfilm during the initial stages of mineral growth. During incubation ofPLG scaffolds in SBF containing ¹²¹I VEGF, the amount of VEGF measuredin the scaffolds did not change significantly after day 2. The presentstudy limited the time frame for SBF incubation to 5 days, since thiswas a period in which mineral growth was initiated, and significantmineral growth occurred in a previous study on gas foamed/particulateleached 85:15 PLG scaffolds.

[0258] Previous studies on mineralized PLG scaffolds show that mineralgrowth continues for at least two weeks in vitro and it considered thatbioactive factors may incorporate into the mineral film for longerincubation periods. Notably, the attenuation of the release of VEGF fromPLG scaffolds caused by mineral formation cannot be explained byincorporation of the protein into the mineral film, since thisattenuation occurs primarily within the first 5 days of SBF incubation,and there is no significant incorporation during this time period.

[0259] Because the attenuation of growth factor release from PLGscaffolds cannot be explained by incorporation of proteins back into thescaffolds after release, or incorporation of proteins into the growingmineral crystals, it is likely that the attenuation is simply due to abarrier effect. The mineral crystal growing on the inner pore surfacesof the PLG scaffold may physically block the release of proteins fromthe polymer matrix. This barrier effect has been studied extensively incontrolled drug delivery applications using layered polymericmicrospheres and microspheres encapsulated in microporous membranes, andthe growth of bone-like mineral represents a new method for blockingprotein diffusion out of polymeric materials.

[0260] Thus, vascular endothelial growth factor does not incorporateinto the mineral film or significantly incorporate into the polymerscaffold during incubation of PLG in SBF. The previously observed effectof mineral growth on VEGF release from PLG scaffolds is likely caused bythe mineral acting as a physical barrier to protein diffusion out of thescaffold. This mechanism is contemplated to be useful in controlled drugdelivery applications, as the release profile from these materials couldbe predictably controlled by mineral film thickness and density.

[0261] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of certain preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the compositions and methods and in the steps or in the sequence ofsteps of the methods described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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1. canceled
 2. The method of claim 53, wherein said surfacemineralization comprises a patterned mineral layer comprising aplurality of discrete mineral islands.
 3. The method of claim 53,wherein said surface mineralization comprises a substantiallyhomogeneous mineral coating. 4-8. canceled.
 9. The method of claim 53,wherein said biomaterial comprises at least a first portion that is a3-dimensional biomaterial scaffold.
 10. The method of claim 53, whereinsaid biomaterial comprises at least a first portion that is a porous,degradable polymer biomaterial.
 11. The method of claim 10, wherein saidbiomaterial comprises at least a first portion that is a polylactic acid(PLA) polymer, polyglycolic acid (PGA) polymer or polylactic-co-glycolicacid (PLG) copolymer biomaterial.
 12. The method of claim 10 whereinsaid biomaterial comprises at least a first portion that is a porous,degradable polymer biomaterial that has an interconnected pore structureand that is degradable over a controllable time scale.
 13. canceled. 14.The method of claim 93, wherein said processing technique comprises agas foaming and particulate leaching technique. 15-17. canceled.
 18. Themethod of claim 9, wherein said biomaterial comprises at least a firstportion that is a 3-dimensional biomaterial scaffold comprising an innerpore surface and wherein said surface mineralization is generated onsaid inner pore surface.
 19. The method of claim 53, wherein saidsurface mineralization comprises calcium.
 20. The method of claim 53,wherein said surface mineralization comprises a mixture of at least afirst and second mineral. 21-25. canceled.
 26. The method of claim 53,wherein said biomaterial is operatively associated with a biologicallyeffective amount of at least a first bioactive substance or biologicalcell. 27-35. canceled.
 36. The method of claim 26, wherein said at leasta first bioactive substance or biological cell is incorporated into saidbiomaterial prior to executing said at least a first surfacemineralization process.
 37. The method of claim 26 wherein said at leasta first bioactive substance or biological cell is incorporated into saidbiomaterial during said at least a first surface mineralization process.38. The method of claim 26, wherein said at least a first bioactivesubstance or biological cell is incorporated into said biomaterialsubsequent to said at least a first surface mineralization process. 39.The method of claim 26, wherein the generation of said surfacemineralization controls the release of said bioactive substance orbiological cell from said biomaterial. 40-43. canceled.
 44. The methodof claim 26, wherein said biomaterial is operatively associated with abiologically effective amount of at least a first mineral-adherent celland wherein the generation of said surface mineralization controls theassociation of said mineral-adherent cell with said biomaterial. 45-52.canceled.
 53. A method for controlling the surface mineralization of abiomaterial polymer, comprising altering the polymer composition orsurface properties of said biomaterial polymer prior to executing atleast a first surface mineralization process.
 54. The method of claim89, wherein controlled surface defects are provided to said biomaterialpolymer to provide a controlled nucleation of discrete mineral islandsat the surface of said biomaterial polymer during said surfacemineralization process.
 55. The method of claim 92, wherein saidbiomaterial polymer is a polylactic-co-glycolic acid copolymerbiomaterial and wherein the ratio of lactide and glycolide componentswithin said copolymer composition is altered. 56-88. canceled
 89. Themethod of claim 53, comprising altering the surface properties of saidbiomaterial polymer prior to executing at least a first surfacemineralization process.
 90. The method of claim 53, comprising alteringthe polymer composition of said biomaterial polymer prior to executingat least a first surface mineralization process.
 91. The method of claim90, wherein the molecular weight of said biomaterial polymer is altered.92. The method of claim 90, wherein the ratio of components within saidbiomaterial polymer is altered.
 93. The method of claim 90, wherein theprocessing technique used to prepare said biomaterial polymer isaltered.
 94. The method of claim 93, wherein said processing techniquecomprises a solvent casting processing technique.
 95. The method ofclaim 93, wherein said processing technique comprises a heat pressingprocessing technique.
 96. The method of claim 93, wherein saidprocessing technique comprises a gas foaming processing technique. 97.The method of claim 53, comprising altering the incubation time of saidat least a first surface mineralization process.
 98. The method of claim53, comprising altering the pH, ionic concentrations or temperaturesused in said at least a first surface mineralization process.
 99. Amethod for controlling the surface mineralization of a gasfoaming/particulate leaching biomaterial, comprising altering thepolymer composition or surface properties of said gasfoaming/particulate leaching biomaterial and executing at least a firstsurface mineralization process; wherein said gas foaming/particulateleaching biomaterial comprises at least a first portion that is preparedby a process comprising gas foaming and particulate leaching.